A field-deployable platform for rapid DNA isothermal amplification and lateral flow detection of fish pathogens.

Effective disease management is essential to preserve ecosystem balance and mitigate mortality in fisheries and aquaculture. However, laboratory requirements, including infrastructure, trained personnel, and long turnaround times, constrain current diagnostic protocols. DNA-based assays offer rapid, field-deployable alternatives, but reliance on single-target analysis and challenges with system miniaturization limit their implementation. We developed and validated an integrated lab-on-a-box platform combining duplex recombinase polymerase amplification with a lateral flow assay (duplex RPA-LFA) for detecting fish pathogens. The assay targets a conserved genus-level region (rRNA gene) for broad screening and species-specific sequences for tailored identification. The complete workflow is performed within a disposable chip that integrates in-situ amplification at 37 °C via a miniaturized heater, followed by detection using an embedded lateral flow strip and a smartphone-based readout. The duplex RPA-LFA was applied for the identification of blood flukes (Cardicola spp.) in farmed tuna and the simultaneous discrimination of C. opisthorchis, C. orientalis, and C. forsteri. The detection limit is 10 DNA copies per reaction, comparable to end-point PCR, with a total runtime of 35 min. Validation across tuna tissues (gill and heart) and surrounding water confirmed reliable detection, including at low parasite loads, supporting early-stage diagnosis without cross-reactivity against non-target tuna pathogens. This platform provides a powerful, user-friendly tool for rapid, on-site pathogen surveillance, enabling timely management decisions to control parasitic outbreaks in marine and aquaculture systems.